For the examination of biological specimens, it has been usual for some time to prepare the specimen with optical markers, in particular with fluorescent dyes. Often, for example in the field of genetic investigations, several different fluorescent dyes are introduced into the specimen and become attached specifically to certain specimen constituents. From the fluorescence properties of the prepared specimen it is possible, for example, to draw conclusions regarding the nature and composition of the specimen, or the concentrations of specific substances within the specimen.
In scanning microscopy, a specimen is illuminated with a light beam in order to observe the detected light emitted, as reflected or fluorescent light, from the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the detected light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam. A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. This detection arrangement is called a “descan” arrangement. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers. Commercial scanning microscopes usually comprise a scanning module that is flange-mounted onto the stand of a conventional light microscope, the scanning module additionally containing all the aforesaid elements necessary for scanning a specimen.
In confocal scanning microscopy, a detection pinhole can be dispensed with in the case of two-photon (or multi-photon) excitation, since the excitation probability depends on the square of the photon density and thus on the square of the illuminating light intensity, which of course is much greater at the focus than in the adjacent regions. The fluorescent light being detected therefore very probably originates almost exclusively from the focus region, which renders superfluous any further differentiation, using a pinhole arrangement, between fluorescent photons from the focus region and fluorescent photons from the adjacent regions.
Many fluorescent dyes can be excited only with ultraviolet illuminating light. The use of ultraviolet illuminating light has the disadvantage, especially for living specimens, of much greater specimen damage. In addition, all optical components must be transparent to ultraviolet light and to the fluorescent light that, because of the Stokes shift, has a greater wavelength, and must not be damaged as a result of illumination with ultraviolet light. With cemented optical components in particular, such as lens element groups in a microscope objective, illumination with ultraviolet light causes irreversible damage to the cement and to the lens elements. A further disadvantage of illumination with ultraviolet light has to do with the shallower penetration depth into biological specimens. The disadvantages can be eliminated by two-photon or multi-photon excitation. In multi-photon scanning microscopy, the fluorescent photons that are attributable to a two-photon or multi-photon excitation process are detected. The probability of a two-photon transition depends on the square of the exciting light power level. In order to achieve high light power levels, it is therefore useful to pulse the illuminating light in order to achieve high peak pulse power levels. This technique is known, and is disclosed e.g. in U.S. Pat. No. 5,034,613 “Two-photon laser microscopy” and in German Unexamined Application DE 44 14 940. A further advantage of multi-photon excitation, especially in confocal scanning microscopy, is improved bleaching behavior, since the specimen bleaches only in the region of sufficient power density, i.e. at the focus of an illuminating light beam. Outside this region, in contrast to one-photon excitation, almost no bleaching takes place.
In a microscope with multi-photon excitation of the specimen, a mode-coupled laser that emits a pulse train with ultra-short pulses is usually used as the light source; the pulses have time-defined pulse widths in the vicinity of a few picoseconds or a few femtoseconds. The repetition rate of the pulses is typically 80 MHz. At present, the time-defined pulse width cannot be directly measured even with the fastest photodiodes. Indirect measurement methods and complex autocorrelators, which are expensive and in most cases usable only for a limited wavelength region, are used to ascertain the time-defined pulse widths. Only the pulse train itself, i.e. the sequence of pulses, is detectable with fast detectors.
A parasitic measurement of the time-defined pulse widths of the exciting light during microscopic examination of a specimen is important, since the pulse widths of the pulses emitted by the laser may fluctuate greatly; this directly influences the nonlinear effects that are being deliberately excited in the specimen (two-photon absorption, generation of second harmonic, etc.), and can thus result in incorrect information in the image. Parasitic monitoring of this kind with an autocorrelator is very complex and laborious.
German Unexamined Application DE 199 19 091 A1 discloses an arrangement for adjusting the laser power level and/or pulse length in a microscope, a specimen being excited by irradiation with a short-pulse laser to produce nonlinear fluorescence, preferably two-photon fluorescence; the nonlinear fluorescence signal, as well as a reflected signal and/or a reference signal corresponding to the laser power level, being detected; and the ratio of the fluorescence signal to the reflected signal and/or reference signal serving as a control signal for adjustment.